1. Field of the Invention
The present invention relates to novel metal-chelator conjugates which comprise a biotin or avidin derivative linked to 1,4,7,10-tetraazacyclododecane-N, N', N", N'"-tetraacetic acid (DOTA). The derivatives are useful, for example, for targeting radiometals to specific sites in vivo using pretargeting protocols for the detection and treatment of tumors, lesions, and sites of infection.
2. Description of Related Art
Metal detection and therapeutic agents can be targeted to in vivo target sites, such as tumors, lesions, and sites of infection, using pretargeting protocols. In pretargeting protocols, a primary targeting species comprising (i) a first targeting moiety which binds to the target site and (ii) a binding site that is available for binding by a subsequently administered second targeting species is targeted to an in vivo target site. Once sufficient accretion of the primary targeting species is achieved, a second targeting species comprising (i) a diagnostic or therapeutic agent and (ii) a second targeting moiety, which recognizes the available binding site of the primary targeting species, is administered. An illustrative example of a pretargeting protocol is the biotin-avidin system for administering a cytotoxic radionuclide to a tumor. In a typical procedure, a monoclonal antibody targeted against a tumor-associated antigen is conjugated to avidin and administered to a patient who has a tumor recognized by the antibody. Then the therapeutic agent, e.g., a chelated radionuclide covalently bound to biotin, is administered. The radionuclide, via its attached biotin is taken up by the antibody-avidin conjugate pretargeted at the tumor. Examples of pre-targeting biotin/avidin protocols are described, for example, in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al. 4, J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl. Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 4 48:167, 1991; Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl. Med. Commun. 12:211, 1991; Stickney et al., Cancer Res. 51:6650, 1991; and Yuan et al., Cancer Res. 51:3119, 1991; all of which are incorporated by reference herein in their entirety.
Three-step pretargeting protocols in which a clearing agent is administered after the first targeting composition has localized at the target site also have been described. The clearing agent binds and removes circulating primary conjugate which is not bound at the target site, and prevents circulating primary targeting species (antibody-avidin or conjugate, for example) from interfering with the targeting of active agent species (biotin-active agent conjugate) at the target site by competing for the binding sites on the active agent-conjugate. When antibody-avidin is used as the primary targeting moiety, excess circulating conjugate can be cleared by injecting a biotinylated polymer such as biotinylated human serum albumin. This type of agent forms a high molecular weight species with the circulating avidin-antibody conjugate which is quickly recognized by the hepatobiliary system and deposited primarily in the liver.
Examples of these protocols are disclosed, e.g., in Axworthy et al., PCT Application No. WO 93/25240; Paganelli et al., "Monoclonal Antibody Pretargeting Techniques For Tumour Localization: The Avidin-Biotin System", Nucl. Med. Comm., Vol. 12:211-234, (1991); Oehr et al., "Streptavidin And Biotin As Potential Tumor Imaging Agents", J. Nucl. Med., Vol. 29:728-729, (1988); Kalofonos et al., "Imaging Of Tumor In Patients With Indium-111-Labeled Biotin And Streptavidin-Conjugated Antibodies: Preliminary Communication", J. Nucl. Med., Vol 31:1791-1796, (1990); Goodwin et al., "Pre-Targeted Immunoscintigraphy Of Murine Tumors With Indium-111-Labeled Bifunctional Haptens", J. Nucl. Med., Vol. 29:226-234, (1988). Improved pretargeting protocols using the biotin-avidin system are disclosed, e.g., in our U.S. Pat. Nos. 5,525,338 and 5,482,698 and co-pending U.S. patent applications Ser. Nos. 08/486,166 and 08/731,107, the disclosures of which are incorporated by reference herein in their entirety.
Many of the above-described pretargeting protocols use conjugates comprising biotin and a chelated metal to deliver metal detection and/or therapeutic agents to target sites. One chelating agent, 1,4,7,10-tetraazacyclododecane-N, N, N", N'"-tetraacetic acid (DOTA), is of particular interest because of its ability to chelate a number of diagnostically and therapeutically important metals, such as radionuclides and radiolabels.
Conjugates of DOTA and proteins such as antibodies have been described. For example, U.S. Pat. No. 5,428,156 teaches a method for conjugating DOTA to antibodies and antibody fragments. To make these conjugates, one carboxylic acid group of DOTA is converted to an active ester which can react with an amine or sulfhydryl group on the antibody or antibody fragment. Lewis et al., Bioconjugate Chem. 5: 565-76 (1994), describes a similar method wherein one carboxyl group of DOTA is converted to an active ester, and the activated DOTA is mixed with an antibody, linking the antibody to DOTA via the .epsilon.-amino group of a lysine residue of the antibody, thereby converting one carboxyl group of DOTA to an amide moiety.
Conjugates of DOTA and biotin have been described, but many of these require complicated synthesis steps or are not stable in vivo. For example, Su, J. Nucl. Med., 36 (5 Suppl):154P (1995), discloses the linkage of DOTA to biotin via available amino side chain biotin derivatives such as DOTA-LC-biotin or DOTA-benzyl-4-(6-amino-caproamide)-biotin. However, these biotin derivatives have been shown to be unstable in blood serum.
Yau et al., WO 95/15335, disclose a method of producing nitro-benzyl-DOTA compounds which can be conjugated to biotin. The method comprises a cyclization reaction via transient projection of a hydroxy group; tosylation of an amine; deprotection of the transiently protected hydroxy group; tosylation of the deprotected hydroxy group; and intramolecular tosylate cyclization. This reaction scheme is very lengthy and complicated and the resulting product comprises at least one chiral carbon and may suffer from such problems as, unequal stability and reactivity of enantiomers.
Wu et al., Nucl. Med. Biol., Vol. 19 (2):239-44 (1992), discloses a synthesis of macrocylic chelating agents for radiolabeling proteins with .sup.111 IN and .sup.90 Y. Wu et al. makes a labeled DOTA-biotin conjugate to study the stability and biodistribution of conjugates with avidin, a model protein for studies. This conjugate was made using a biotin hydrazide which contained a free amino group to react with an in situ generated activated DOTA derivative. Although the DOTA-biotin-avidin conjugates were found to be stable, the resultant .sup.90 Y-labeled and the resultant .sup.111 In labeled compounds achieved only 12% and 29% binding under avidin, respectively, even when incubated with excess avidin.
There is a need, therefore, for a DOTA-biotin conjugate suitable for in vivo use that is stable, readily synthesized, and that can bind avidin efficiently.